转录因子FOXO3对人胚胎干细胞心肌定向分化的调控作用与机理的研究

Heart failure remains one of the leading causes of morbidity and mortality throughout the world. With the advances in stem cell biology, it is possible to replace the injured cardiac tissue and this cell-based therapy keeps the hope of a potential cure for heart failure. However, after a decade of efforts in pre-clinical and clinical research, cell therapy still cannot be considered as a routine therapy as pharmaceutical treatment for heart failure patients because of the poor understanding of the molecular mechanisms of regulation of cardiac differentiation. Studies with transgenic mice indicate an important role for FoxO transcription factors in mouse heart development but there is no evidence to date for the role of FoxO in the regulation of human cardiogenesis. Published data shows that WNT signaling pathway and ROS are key regulators for cardiac differentiation. FoxOs have been proved to be able to interact with WNT and ROS to regulate the cell function in different context. Our previous results show that the expression of FOXO3 undergoes a 2.5-fold upregulation and it is transcriptionally activated during the cardiac differentiation from human embryonic stem cell (hESC). Based on these results, we hypothesized that FOXO3 interacts with WNT signaling pathway in the presence of ROS and activates the expression of target genes to regulate the cardiac differentiation. To prove this hypothesis, loss-of-function and gain-of-function strategy will be used during the cardiac differentiation of hESC to investigate the role of FOXO3 in the regulation of cardiac differentiation process and efficiency. Then, the interaction and relationship between FOXO3, WNT signaling and ROS will be investigated using co-Immunoprecipitaton,ROS scavenger treatment and other methods to further demonstrate the molecular mechanisms of regulation of cardiac differentiation by FOXO3. The increasing knowledge and better understanding of the mechanisms of FOXO3 in the regulation of cardiac differentiation will provide us new ideas and targets for drug screening and therapeutic method development to improve the cardiac differentiation efficiency and finally boost biotechnological resources closer to clinical translation.

转基因动物研究显示FoxO家族成员对小鼠的心脏发育具有重要调控作用，但尚无其参与人心脏发育调控的直接证据。FoxO可通过与WNT通路及ROS的相互作用调节细胞功能，而后两者已被证实对心肌分化至关重要。我们的前期研究发现：FOXO3在人心肌分化过程中表达升高且处于转录活化状态。我们据此推测：FOXO3可在ROS参与下与WNT通路相互作用，启动相关靶基因的表达而调控人心肌细胞的分化。为证明此假说，本课题拟在诱导人胚胎干细胞向心肌分化过程中采用功能缺失与功能获得两种策略，观察FOXO3对人心肌分化进程及效率的影响；然后以免疫共沉淀、药物干预ROS水平等手段，研究心肌分化过程中FOXO3、WNT通路及ROS间的相互作用，进一步揭示FOXO3参与人心肌分化调控的分子机制。本研究将从一个新的视角丰富对人心肌分化调控网络的认识，为提高心肌诱导分化效率用于修复病损心肌，根治心衰提供新的思路与药物作用靶点。

为进一步阐明心肌分化的分子调控机制，以优化改进诱导方法，提高心肌细胞的体外诱导分化效率及纯度，使之尽早用于心血管疾病的临床治疗。我们在前期研究基础上，以FOXO3a为切入点，希望阐明其在人胚胎干细胞向中胚层及心肌分化过程中的调控作用与机理。我们首先采用功能缺失策略，通过慢病毒介导的FOXO3a特异性shRNA建立FOXO3a敲低的人胚胎干细胞系HES2/shFOXO3,观察其对人心肌分化进程及效率的影响；然后观察药物干预内源性ROS水平对分化的影响，同时通过免疫共沉淀等手段，研究心肌分化过程中FOXO3a、WNT通路及ROS间的相互作用.结果显示，1. FOXO3a在人胚胎干细胞心肌定向分化过程中表达升高且由未分化状态下的细胞质进入细胞核内，显示其活化状态。2. FOXO3a表达抑制使中胚层分化相关标记基因如T、MESP2、HAND2等表达水平提高或峰值提前，暗示可能促进了中胚层的分化；但随后抑制了心肌分化标记基因的表达并使心肌分化末期肌钙蛋白T（TroponinT）阳性细胞的比例明显降低，显然抑制了心肌分化的进程与效率。3. FOXO3a敲低后，细胞内源性ROS水平明显升高，提示FOXO3a可能通过调节细胞内ROS水平参与了人胚胎干细胞中胚层及心肌定向分化的调控。4. 人胚胎干细胞心肌定向分化早期，细胞内源性ROS水平的升高对于中胚层与生心中胚层分化至关重要，通过干预细胞内ROS水平可明显影响中胚层及心肌分化的进程和效率。5.在人胚胎干细胞心肌定向分化过程中，使用JNK抑制剂SP600125处理细胞后，14-3-3蛋白磷酸化水平降低，FOXO3a总蛋白水平显著降低且入核减少，与对照组相比心肌诱导分化进程明显受到抑制且所得心肌细胞数量明显减少。6.免疫共沉淀结果显示，在人胚胎干细胞心肌定向分化过程中，FOXO3a入核并且与β-Cartenin/TCF-1形成复合物调控下游靶基因的表达。.基于本研究的结果，可通过调控ROS水平优化体外干细胞心肌诱导分化方案，缩短诱导进程，提高人胚胎干细胞体外诱导心肌分化的效率和细胞产率。此外，在人胚胎干细胞心肌定向分化过程中，可能通过调节JNK通路活性，调节心肌细胞分化与增殖，提高心肌细胞产出效率。